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PDPN (podoplanin) gene targeted humanized chimeric antigen receptor and application thereof

A chimeric antigen receptor and gene technology, applied in the field of immunology, can solve problems affecting the killing effect of PDPN-positive tumor cells, and achieve the effect of avoiding HAMA

Inactive Publication Date: 2017-09-22
SHANGHAI SHENYIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the murine scFv will produce host anti-graft reaction (HAMA) after infusion into the human body, which seriously affects the killing effect of modified T cells on PDPN-positive tumor cells.

Method used

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  • PDPN (podoplanin) gene targeted humanized chimeric antigen receptor and application thereof
  • PDPN (podoplanin) gene targeted humanized chimeric antigen receptor and application thereof
  • PDPN (podoplanin) gene targeted humanized chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Obtaining of chimeric antigen receptor hPDPN single-chain antibody

[0039] Humanized murine antibody was designed by means of bioinformatics to obtain the coding nucleotide sequence of the humanized antibody, the single-chain antibody scFv was purified, and experimental verification was carried out, as follows:

[0040] 1. Design humanized antibody heavy chain and light chain sequences

[0041] According to the existing Anti-Podoplanin murine antibody sequence, the humanized heavy chain and light chain of the antibody were constructed by combining the amino acids in the CDR region of the heavy chain and the light chain with the human framework respectively by using the framework replacement and random mutation scheme Sequence, the amino acid sequence of the obtained humanized antibody heavy chain is SEQ ID NO.1, the nucleic acid sequence is SEQ ID NO.2; the amino acid sequence of the humanized antibody light chain is SEQ ID NO.3, the humanized light chain Th...

Embodiment 2

[0077] Example 2 Construction of chimeric antigen receptor anti-human PDPNscFV-CD137-CD3ζ lentiviral plasmid

[0078] According to the humanized single-chain antibody scFv sequence information obtained in Example 1, construct the PDPNscFv-CD137-CD3ζ lentiviral expression vector, and prepare the vector for lentiviral production, including the following steps:

[0079] According to the results of FACS, the single-chain antibody sequence capable of binding to the target protein was selected as a template to construct an anti-human hPDPN chimeric antigen receptor (hPDPN-CAR, referred to as CAR, the same below), and the T cell stimulation signal transduction structure was based on human The domain composed of the hinge region of CD137 and the intracellular region of human CD3ζ in series, its amino acid sequence is shown in SEQ ID NO.9, its nucleotide sequence is shown in SEQ ID NO.10, and the structure of the obtained hPDPN-CAR is schematically shown in figure 1 Its amino acid sequ...

Embodiment 3

[0084] Packaging, concentration and titer determination of embodiment 3 lentivirus

[0085] Production of hPDPNscFV-CD137-CD3ζ lentivirus in preparation for preparation of recombinant CAR-T cells.

[0086] 1. Preparation of lentivirus

[0087] Prepare a 15cm cell culture dish and inoculate 5*10 6 cells / dish, add complete medium (DMEM high glucose, 10% FBS), place at 37°C, 5% CO 2 incubator, overnight.

[0088] Take out the PEI and lentiviral packaging plasmid (CAR lentiviral expression vector and lentiviral packaging helper plasmid) from the refrigerator, thaw at room temperature, and mix completely by blowing up and down with a pipette gun. Remove the PBS or HBSS buffer and warm to room temperature. Take 2 mL of LPBS to one well of a 6-well plate, add 10 μg Lenti-EF1a-CAR and 11 μg Lenti-Mix respectively, mix well by pipetting up and down, then add 26 μL of 100 μM PEI, immediately pipette up and down to mix, room temperature Let stand for 10 minutes.

[0089] Add the ab...

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Abstract

A human chimeric antigen receptor targeting PDPN gene and its use, which contains a single-chain antibody hPDPNscFV against human PDPN antigen and a T cell stimulating signal transduction structure, wherein the light chain of the single-chain antibody hPDPNscFV can be The variable region and the heavy chain variable region are connected by a connecting peptide, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.5, the scFV in the chimeric antigen receptor has the specificity of anti-human PDPN, and can specifically The chimeric antigen receptor can be used to modify T lymphocytes, and the modified T cells can be used for the treatment of PDPN-positive tumors, especially for surface tumors. PDPN-positive glioma cells have a specific killing effect.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a human chimeric antigen receptor targeting PDPN gene and its application. Background technique [0002] Designing autologous antigen-specific T cells generated in vitro to mediate immune responses through tumor-specific T cells is a promising approach to treat tumors. At present, it has been confirmed that the isolation and transfer of tumor-specific T cells has achieved good clinical results in the treatment of melanoma and hematological tumors. [0003] Chimeric antigen receptor (CAR, chimeric antigen receptor) obtains novel specificity of T cells through exogenous gene transduction technology. As a synthetic receptor, CAR is composed of a scFV (single-chain antibody, composed of the amino acid sequence of the VL region of the antibody and the amino acid sequence of the VH region of the antibody connected by a Linker) that can recognize tumor-associated specific antigens...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/63A61K39/395A61P35/00
CPCC07K16/3092A61K2039/505C07K14/7051C07K14/70596C07K16/36C07K2317/24C07K2317/622C07K2319/00
Inventor 朱剑虹钟俊杰王智富汤奇胜
Owner SHANGHAI SHENYIN BIOTECH
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